Glycosphingolipids are an important class of glycolipids and can be regarded as glycosides of N-acylsphingosine. These glycosphingolipids are generally divided into three classes: (1) neutral glycolipids. (2) sulfatides (sulfate-containing glycolipids), and (3) gangliosides (sialic acid- (N-acyl or O-acyl neuraminic acid) containing glycolipids).
Glycosphingolipids are also classified according to the number of sugar residues and according to the oligosaccharide core structures.
Besides gangliosides three series of neutral glycolipids are known, i.e., lacto, globo, and ganglio series. In addition, the simplest glycosphingolipids, containing only one carbohydrate, are known as cerebrosides. Currently, approximately 130 molecular species of glycosphingolipids are known, many of which are present at the cell surface membrane (Hakomori, S. (1986) Scientific American 254, 44-53).
Glycosphingolipids and, in particular, gangliosides are of great interest due to their activity as modifiers of cell physiology (E. Bremer, et al. (1986) J. Biol. Chem. 261, 2434-2440: S. Hakomori, et al., in G. Tettamanti, R. Ledeen, Y. Nagai, K. Sandhoff, and G. Toffano (Eds.), Neuronal plasticity and gangliosides, Liviana Press, Pavoda, 1986 pp. 201-214: and N. Hanai et al., (1987) Biochem. Biochys. Res. Commun., 147, 127-134). Therefore, the synthesis of various glycosphingolipid derivatives and analogues is an important area of research.
Cell surface gangliosides may have two basic cellular functions: i) to mediate cell social functions (cell-cell, cell-microbe, or cell-matrix interactions), and ii) to modulate functional membrane proteins such as receptors and transporters (Hakomori, S (1981) Ann. Rev. Biochem. 50, 733-764). Ganglioside-mediated modulation of membrane receptor function has been suggested by the modification of protein kinase activity of epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors by specific gangliosides (GM.sub.3 or GM.sub.1), but not by other types of glycolipids (Bremer, E., et al (1984) J. Biol. Chem. 259, 6818-6825 and Bremer, E., et al (1986) J. Biol. Chem. 261, 2434-2440).
For example, in A431 cells, which are characterized by a high content of EGF receptor, tyrosine phosphorylation of EGF receptors was specifically inhibited by exogenous addition of GM.sub.3 but not by other gangliosides or neutral glycolipids (Bremer, E., et al (1986) J. Biol. Chem. 261, 2434-2440). Inhibition of the receptor kinase was demonstrated on the isolated EGF receptor after adsorption on an anti-receptor-antibody-Sepharose complex (Bremer, E., et al (1986) J. Biol. Chem. 261, 2434-2440). Further, exogenous addition of these gangliosides. GM.sub.3 and GM.sub.1, affects fibroblast growth factor- (FGF), EGF-, or PDGF-dependent cell growth stimulation in chemically-defined media (Bremer, E. et al (1986) J. Biol. Chem. 261, 2434-2440; Bremer, E., and Hakomori, S. (1982) Biochem. Biophys. Res. Commun. 106, 711-718: and Hakomori, S., et al (1986) in Neuronal plasticity and gangliosides (Tettamanti, G., Ledeen, R., Nagai, Y., Sandhoff, K., and Toffano, G., eds.), pp. 201-214. Liviana Press, Padova, Italy).
More recently, cytoskeletal protein kinase (Tsuji, S., et al (1983) J. Biochem. (Tokyo) 94, 303-306) has been found to be modified by gangliosides and various cell growth modulators such as retinoids, butyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) have been reported to induce changes in ganglioside synthesis when cell growth is arrested (Patt. L., et al (1978) Nature 273, 379-381; Fishman, P. H., et al (1974) Biochem. Biochys. Res. Commun. 59, 292-299; Huberman, E., et al (1979) Cancer Res. 39, 2618-2624; and Burczak, J. D. et al. (1983) Exp. Cell Res. 147, 281-286). On the other hand, sphingosines have been shown to have a non-specific common inhibitory effect on protein kinase C activity (Hannun, Y. A., et al (1986) J. Biol. Chem. 261, 12604-12609).
Recently, various gangliosides (GM.sub.3, GM.sub.2, GM.sub.1, and GD.sub.1a) have been derivatized (S. Neuenhofer, G. et al (1985) Biochemistry, 24, 525-532) into their lyso forms, in which the amino groups of neuraminic acid and hexosamines were N-acetylated and only the amino group of the sphingosine was unsubstituted. For this derivatization, the amino group of the sphingosine was first blocked by a hydrophobic protective group (9-fluorenylmethoxycarbonyl), followed by acetylation of the amino groups of neuraminic acid and hexosamines, and subsequent removal of the protective group by liquid ammonia. The procedure involves several steps, and the yield is poor (about 30%).
The lyso form of GM.sub.3 has been prepared (T. Taketomi and N. Kawamura, (1970) J. Biochem. (Tokyo) 68, 475-485) by treatment of GM.sub.3 with a refluxing solution of 1M KOH in aqueous 90% butanol for 2.5 h. The product was claimed to have a strong hemolytic activity (twice that of lysolecithin) and was called lysosphingolipid or lysohematoside. The compound, however, had free amino groups at both neuraminic acid and sphingosine. (The term "neuraminic acid" is used according to the original definition (G. Blix, et al., (1957) Nature 179, 1088), i.e., de-N-acyl sialic acid is defined as "neuraminic acid", while N-acetyl, N-glycolyl and O-acyl derivatives of neuraminic acid are collectively called "sialic acid.")
Because of the remarkable cell growth modifying activities of gangliosides, potential application of gangliosides to modulation of in vitro and in vivo cell growth is an exciting area for investigation,
Further, because gangliosides occur naturally in many cells, use of inhibitors or promoters of synthesis of particular gangliosides to abolish or enhance the natural effects of the gangliosides is also an exciting area for investigation. Unfortunately, because of the unpredictable cell growth modifying activities of different gangliosides which have been studied, e.g. GM.sub.3 and GM.sub.1, and because of lack of any coherent information as to what types of cells react to each ganglioside, little progress has been made in the area of in vitro or in vivo applications of gangliosides to modulate cell growth.
Accordingly, it would be desirable to be able to identify gangliosides that have the same effects on a variety of cells so that concrete in vitro or in vivo applications of the gangliosides can be identified and used for practical purposes.